This work was supported by a grant to MEM from the Dept. of Life Sciences and Systems Biology of the University of Turin, Italy. The authors thank G. Gnavi for technical assistance during some experiments. CTR is funded by the Wellcome Trust and the Royal Society (Grant Number 098436/Z/12/Z).

1. Setting of the Triaxial Coils
NOTE: Figure 1 shows the triaxial coils used to reverse the GMF.
<ol>
	<li>Turn on the three-axis magnetometer, whose probe is inserted in the triaxial coils.</li>
	<li>Turn on the computer and launch the magnetometer software that allows data to be collected from three-axis magnetometer.</li>
	<li>Use the component values reported by the magnetometer to calculate the magnitude of the GMF. For instance, with magnetometer values: Bx = 6.39 &#181;T, By = 36.08 &#181;T, Bz = 20.40 &#181;T calculate a field strength of 41.94 &#181;T by using the following equation: B = BGMF + Badditional, where Badditional = (Bx2 + By2 + Bz2)&#189; (i.e., 41.9 &#181;T in the example.)</li>
	<li>Turn on the three DC power supplies (dual range: 0-8V/5A and 0-20V/2.5A, 50W) each one connected to three couples of Helmholtz coils and connected to a computer via a GPIB connection (Figure 1B).</li>
	<li>Set voltages of the power supplies to generate the desired magnetic field with a reversed magnetic field vector. For instance, with BGMF as in step 1.3 and with the coil size of the instrumentation described here, set the voltages to Vx =0.00 V, Vy = 30.52 V, Vz = 0.00 V in order to generate a new resultant B = BGMF + Btriaxial coils = (6.38, -36.08, 20.39) &#181;T. i.e., a new field with the same magnitude as BGMF but pointing to a different direction.</li>
	<li>Verify the new field with the magnetometer software by using the procedure described in 1.3.</li>
	<li>Expose plants to both normal and reversed GMF conditions by using Petri plates as described in section 2.</li>
	<li>Perform sham exposure experiments by keeping the magnitude of the field equal to | BGMF| and keeping the vertical component of the field equal to that of the GMF but altering the direction (i.e., &#8220;North, East or West&#8221;) of the horizontal component of the field with equal currents in the triaxial coils compared to the field reversal condition. Do this by altering the voltage of the coils as described in 1.5. 
	Note: This sham exposure rules out potential subtle heating or vibrational effects from either the coils themselves or from the electronics used to control the coils.</li>
	<li>Run double-blind experiments by applying field conditions blinded from the personnel performing the remainder of the experiments and/or interpreting the data.</li>
</ol>
2. Preparation of Plant Materials and Conditions for Plant Growth
<ol>
	<li>Use seeds of Arabidopsis thaliana, ecotype Columbia 0 (Col 0), place then into a 1.5 ml tube and surface sterilize by treatment with a 5% (w/v) calcium hypochlorite solution and 0.02% (v/v) Triton X-100 in 80% ethanol (EtOH), for 10-12 min at 25-28 &#176;C, with continuous shaking. Then rinse twice with 80% EtOH, wash with 100% EtOH and finally rinse with sterile distilled water.</li>
	<li>Prepare 1 L of Murashige and Skoog 22 (MS) modified medium by adding: 2.297 g of MS (0.5 x MS Basal Salt Mixture), 10 g Sucrose, deionized water up to 1 L, pH 5.8-6.0 adjusted with KOH. Add 16 g of agar and autoclave for 20 min, 120 &#176;C.</li>
	<li>Before solidification, pour 80 ml of medium into each (120 x 120 mm2) square Petri plates. Sow thirty sterile seeds on plate, and then seal plates with a waxy film.</li>
	<li>Vernalize the plates horizontally in darkness at 4 &#176;C for 2 days to potentiate and synchronize germination, and then expose Petri plates to either normal or reversed GMF.</li>
	<li>Expose seeds in a climate controlled environment at 22 &#176;C in a vertical position in parallel experiments both inside the triaxial coils and outside the triaxial coils under a photoperiod schedule of 8 hr darkness and 16 hr light, using Sodium vapor lamps (220 x 10-6 E m-2s-1). Use a blue gelatin film for spotlight to reduce the red component of lamps.</li>
	<li>Expose plants for 10 days before RNA extraction to both normal (control) and reversal (treatment) GMF conditions.</li>
	<li>After exposure, take pictures of Petri dishes.</li>
	<li>Use the ImageJ software to calculate root length and leaf areas.
	<ol>
		<li>Briefly, measure the side of the Petri plate, then open the image of the Petri plate and by using the &#8220;straight&#8221; line option to draw a line that exactly crosses the plate side.</li>
		<li>In the &#8220;analyze&#8221; menu select &#8220;set scale&#8221; and insert the actual distance in the &#8220;known distance&#8221; box (e.g., 120 mm), then insert the unit of length (mm); finally click the &#8220;global&#8221; option to make settings available for all measurements.</li>
		<li>For root length, follow carefully the shape of the root by using the freehand tool. Measure the length by using the &#8220;measure&#8221; option in the &#8220;analyze&#8221; menu. Continue to measure all roots in the picture and save the file for further statistical analyses.</li>
		<li>For leaf area, from the &#8220;image&#8221; menu use the adjust option and then &#8220;color threshold&#8221;. Select the individual leaf and in the &#8220;analyze&#8221; menu select &#8220;analyze particles&#8221;. Save the individual measurements for statistical analyses.</li>
	</ol>
	</li>
</ol>
3. Arabidopsis Total RNA Extraction, Quantitative Real Time-PCR (qPCR) Reaction Conditions and Primers for Arabidopsis
<ol>
	<li>Collect separately 30 shoots and 30 roots and immediately freeze in liquid nitrogen. Then grind in liquid nitrogen with mortar and pestle.</li>
	<li>Isolate total RNA using a purification kit and RNase-Free DNase treatment kit by using manufacturer&#8217;s instructions.</li>
	<li>Check sample quality and quantity by using an RNA nano kit and capillary gel electrophoresis according to manufacturer&#8217;s instructions. Confirm the quantification of RNA spectrophotometrically.</li>
	<li>Use 2 &#181;g of total RNA and random primers using a cDNA Reverse Transcription Kit to obtain first strand cDNAs according to the manufacturer&#8217;s recommendations.</li>
	<li>Perform all experiments on a Real-Time System using SYBR green I with ROX as an internal loading standard.</li>
	<li>Perform the reaction with 25 &#181;l of mixture consisting of 12.5 &#181;l of 2x SYBR Green qPCR Master Mix, 0.5 &#181;l of cDNA and 100 nM primers. Use the primers listed in Table 1. Include in controls non-RT controls (using total RNA without reverse transcription to monitor for genomic DNA contamination) and non-template controls (water instead of template).</li>
	<li>Calculate primer efficiency for all primers pairs using the standard curve method 23.</li>
	<li>Use the following PCR conditions: CRU3, COTP1, RRTF1. 10 min at 95 &#176;C, 40 cycles of 15 s at 95 &#176;C, 30 sec at 58 &#176;C, and 30 sec at 72 &#176;C; UBP6, eEF1Balpha2, ACT1, GAPC2, CAT3, TAPX, APX1, RbohD, FeSOD1 10 min at 95 &#176;C, 40 cycles of 15 s at 95 &#176;C, 20 sec at 57 &#176;C, and 30 sec at 72 &#176;C.</li>
	<li>Read fluorescence following each annealing and extension phase. For all runs, perform a melting curve analysis from 55 to 95 &#176;C by including the dissociation segment in the thermal profile. Use the dissociation curve screen, accessed through the results tab to view the dissociation profile (plot of the fluorescence as a function of temperature). Ensure that the dataset collected during the dissociation segment of the experiment is selected for analysis using the Analysis/Setup screen.</li>
	<li>Determine the linear range of template concentration to threshold cycle value (Ct value) by performing a tenfold dilution series (1- to 103-fold) using cDNA from three independent RNA extractions analyzed in three technical replicates 24,25.</li>
	<li>Analyze all amplification plots with the Real-Time PCR instrument software to obtain Ct values. Calibrate and normalize relative RNA levels with the level of the best housekeeping genes as follows: 3.11.1) Access the Amplification Plots screen through the Results tab, select the ramp or plateau for which data should be analyzed using the Analysis Selection/Setup screen, and then select the dRn (baseline-corrected normalized fluorescence) from the Fluorescence menu on the command panel. Access the Plate Sample Values screen through the Results tab to display Ct values for the sampled wells.</li>
	<li>Use four different reference genes [e.g., cytoplasmic glyceraldehyde-3-phosphate dehydrogenase, (GAPC2), ubiquitin specific protease 6 (UBP6), Actin1 (ACT1) and the elongation factor 1B alpha-subunit 2 (eEF1Balpha2)] to normalize the results of the real time PCR. Select the top ranked gene using an analysis software 26; and use the most stable gene for normalization.
	<ol>
		<li>Briefly, organize the input data on an Excel sheet with the first column containing the gene names and first row containing the sample names. Then select the analysis software from the menu-bar. Use the dialog box to select the input data.</li>
		<li>Next, check the fields Sample names, gene names and simple output only. Click on Go button to perform the analysis. Select the top ranked gene (which has the smallest stability value) as candidate gene most stably expressed.</li>
	</ol>
	</li>
	<li>Plot data by showing the differential fold change expression in both shoots and roots.</li>
</ol>
4. Statistical Analyses
<ol>
	<li>Express data as mean values &#177; standard error. Compare the control and the treatment groups by performing analysis of variance (ANOVA) and Tukey&#8217;s test with Bonferroni and Dunn-Sidak Adjusted Probability test (0.95% confidence).</li>
</ol>

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The authors have nothing to disclose.

We recently showed that an amazing correlation exists between GMF reversals and the time when diversion of most of the familial Angiosperm lineages occurred 2. However, despite the stimulating hypotheses and the plethora of studies on the effect of varied GMF intensities, the assumption that GMF reversal may itself induce significant changes in plant gene expression and morphology has never been demonstrated. Here we show for the first time, a method that uses a triaxial octagonal Helmholtz coil to reverse GMF in our laboratory, and that the reversal of the ambient magnetic field can cause phenotypic changes and modulation of gene expression in plants.
In order to obtain GMF reversal (or modification) over a sufficient volume for the plant growth experiments (2 x 2 x 2 m3), we built an octagonal Helmholtz coil system. This system is not commercially available (usually Helmholtz coils are ring-shaped and smaller) and the costs for construction were considerable. Importantly, this system delivers robust field modification, with exceptional time-stability and homogeneity in the modified magnetic fields.
The system is designed and built to reduce the value of the GMF to a thousandth of the normal conditions or to reverse any of the three dimensions of the magnetic field. However, the design of the coils does not allow to generate a high magnetic field strength. Therefore, this instrument in the present form is not suitable for experiments designed to evaluate the effect of high magnetic field strength on plants or other organisms.
In laboratory, alteration of the GMF similar to those described in this method has been obtained by different methods, including shielding by surrounding the experimental zone by ferromagnetic metal plates with high magnetic permeability, which deviate magnetic fields and concentrate them within the metal itself. The advantage of using Helmholtz coils is that the system allows plants to be exposed to more natural conditions (light, air circulation, etc.), thus making it ideal not only for in vitro studies (as with the use of Petri dishes) but also for in vivo plant growth and development experiments. The dimensions of our system create a space that allows a suppression up to &#60;1/1,000th of the natural GMF throughout a 25 x 25 x 25 cm3 spherical volume (see Figure 1A), thus allowing to host several Petri plates or some small pots for plant growth.
The method presented here has been applied to plant biology studies; however, the system allows a wide range of experimentation, including virology and microbiology, as well as studies on nematodes (e.g., Caenorhabditis elegans), arthropods and small animals (including mice and rats). Therefore, tests of the hypothesis that reversal of the GMF is able to induce morphological and transcriptional changes could also be extended to many other living systems, perhaps ultimately even to human cells.
The GMF is constantly changing and fluctuating. Therefore, in our experiments one major challenge is to provide a constant compensation of the GMF in order to obtain the desired new GMF values. This can only be achieved by a continuous control of magnetic fields values through reading of magnetometer values and voltage compensation. Therefore, the system can compensate the slowly varying part of the GMF but it does nothing for higher frequency fluctuations.
In conclusion, the use of triaxial coils to reverse the GMF vector was instrumental to demonstrate that this reversal of the GMF vector is able to induce plant morphological changes and differential gene expression. The results obtained with the presented method provide a compelling evidence in support of the hypothesis that the GMF reversals might have been one of the driving forces for plant evolution over geological timescales 2.

The Earth&#8217;s magnetic field (or equivalently the geomagnetic field, GMF) is an inescapable environmental factor for all organisms living on the planet, including plants. The GMF has always been a natural feature of the Earth, so during evolution, all living organisms experienced its action. An increasing body of evidence shows that the GMF is able to influence many biological processes 1. The GMF is not uniform and there are significant local differences in its magnitude and direction at the surface of the Earth. The GMF at the Earth&#39;s surface shows a broad range of magnitudes, ranging from less than 30 &#181;T to almost 70 &#181;T. The GMF protects the Earth and its biosphere from the lethal effects of solar wind by deflecting most of its charged particles through the magnetosphere2.
Plants respond to environmental stimuli; and classical responses to abiotic factors such as light and gravity have been thoroughly described by defining the so-called phototropic and gravitropic responses. Very little, or nothing, is known on the mechanisms of perception and responses of plants to magnetic fields, despite the plethora of papers published on this topic and recently reviewed 1. Unlike the gravitational field, the GMF changed consistently during plant evolution thereby representing an important abiotic stress factor that has been recently considered a potential driving force eventually contributing to plant diversification and speciation 2. Geomagnetic reversals (or excursions) are changes in polarity of the GMF. During Earth&#8217;s life-history, GMF reversals occurred several times. These exposed the planet to periods of reduced GMF strength during every polarity transition. Some authors have hypothesized that these transitions periods of low GMF strength might have allowed ionizing radiation from the solar wind to reach the Earth&#8217;s surface, thereby inducing a consistent stress to living organisms, which could have been strong enough to induce gene alterations eventually leading to plant evolution 2.
A detailed analysis of experiments describing the effects of magnetic fields on plants shows a large number of conflicting reports, characterized by a dearth of plausible biophysical interaction mechanisms. Many experiments are simply unrealistic, while others lack a testable hypothesis and, ultimately, are unconvincing 3. Over the past years, the progress and status of research on the effect of magnetic fields on plant has been reviewed 2,4-11. Recently, the effect of both low and high magnetic field has been thoroughly discussed 1, with a particular focus on the involvement of GMF reversal events on plant evolution 2.
The most direct means to substantiate the hypothesis that GMF reversals affect plant evolution is to synthesize a GMF reversal in the laboratory by testing the response of plants to normal and reversed magnetic field conditions. To test the hypothesis, we therefore built a triaxial octagonal Helmholtz coil-pairs magnetic field compensation system (triaxial coils), which is able to accurately reverse the normal GMF conditions.
We used Arabidopsis thaliana as a model plant and we tested the effect of reversed GMF on gene expression of some important genes: CRUCIFERIN 3 (CRU3), that encodes a 12S seed storage protein that is tyrosine-phosphorylated and its phosphorylation state is modulated in response to ABA in Arabidopsis thaliana seeds 12,13; the Copper Transport Protein1 (COTP1), that encodes a heavy metal transport/detoxification superfamily protein with the predominant function in soil Cu acquisition and pollen development 14; and Redox Responsive Transcription Factor1 (RRTF1), that encodes a member of the ERF (ethylene response factor) subfamily B-3 of ERF/AP2 transcription factor family that contains one AP2 domain that facilitate the synergistic co-activation of gene expression pathways and confer cross tolerance to abiotic and biotic stresses 15.
Moreover we also analyzed five genes involved in oxidative stress responses: Fe Superoxide Dismutase1, (FSD1), that encodes a cytoplasmic enzyme that enzymatically and rapidly converts the superoxide anion (O2-) and water (H2O) to hydrogen peroxide (H2O2) and molecular oxygen (O2) 16; Catalase3 (CAT3), that that encodes and enzyme that catalyzes the breakdown of H2O2 into water and oxygen 17,18; Thylakoidal Ascorbate Peroxidase (TAPX), that encodes a chloroplastic thylakoid peroxidase that scavenges H2O2 19;Ascorbate Peroxidase1 (APX1), that encodes a cytosolic peroxidase that scavenges H2O2 and represents one of the potential targets of post-translational modifications mediated by NO-derived molecules 20; and NADPH-Respiratory burst oxidase protein D (RbohD) that encodes an enzyme that generates O2- and plays pivotal roles in regulating growth, development and stress responses in Arabidopsis 21.
Our field-reversal methodology provides the first evidence that GMF reversal can induce a significant change in the morphology and gene expression of A. thaliana roots and shoots. This protocol provides an innovative way to evaluate the effect of GMF reversal on plant morphology and gene expression and can be used to assess the potential effect of GMF reversal on other aspects of plant behavior, and thereby guide discussion of the role of GMF reversal on plant evolution.

<table><tbody><tr><td>Three-axis magnetometer</td><td>Bartington&amp;nbsp;</td><td>Mag-03MC</td><td>triaxial fluxgate magnetometer</td></tr><tr><td>Magnetometer power supply</td><td>Bartington&amp;nbsp;</td><td>Mag-03PSU</td><td>triaxial fluxgate magnetometer</td></tr><tr><td>Magnetometer software</td><td>Bartington&amp;nbsp;</td><td>Mag03DAM</td><td>triaxial fluxgate magnetometer</td></tr><tr><td>DC power supply&amp;nbsp;</td><td>Agilent Technologies</td><td>E3642A</td><td></td></tr><tr><td>Calcium hypochlorite&amp;nbsp;</td><td>Sigma</td><td>211389</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma</td><td>X100&amp;nbsp;</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sigma</td><td>2860</td><td></td></tr><tr><td>GroLux Sodium vapor lamps&amp;nbsp;</td><td>OSRAM Sylvania</td><td>600W</td><td></td></tr><tr><td>RNeasy Plant RNA kit&amp;nbsp;</td><td>Qiagen</td><td>74903</td><td></td></tr><tr><td>RNase-Free DNase&amp;nbsp;</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2938B</td><td></td></tr><tr><td>NanoDrop ND-1000&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>not available</td><td></td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit&amp;nbsp;</td><td>Applied Biosystems</td><td>4368813</td><td></td></tr><tr><td>Mx3000P</td><td>Agilent Technologies</td><td>401512</td><td></td></tr><tr><td>2x MaximaTM SYBR Green qPCR Master Mix&amp;nbsp;</td><td>Fermentas International, Inc</td><td>K0221</td><td></td></tr><tr><td>Parafilm</td><td>Sigma</td><td>P7793-1EA</td><td></td></tr><tr><td>Murashige and Skoog Basal Medium</td><td>Sigma</td><td>M5519&amp;nbsp;</td><td></td></tr><tr><td>Petri dish square (120 x120 mm&lt;sup&gt;2&lt;/sup&gt;)</td><td>Sigma</td><td>Z692344&amp;nbsp;</td><td></td></tr></tbody></table>

geomagnetic field (gmf) and plant evolution: investigating the effects of gmf reversal on arabidopsis thaliana development and gene expression

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) and the moment of the radiation of Angiosperms. This led to the hypothesis that alterations in GMF polarity may play a role in plant evolution. Here, we describe a method to test this hypothesis by exposing Arabidopsis thaliana to artificially reversed GMF conditions. We used a three-axis magnetometer and the collected data were used to calculate the magnitude of the GMF. Three DC power supplies were connected to three Helmholtz coil pairs and were controlled by a computer to alter the GMF conditions. Plants grown in Petri plates were exposed to both normal and reversed GMF conditions. Sham exposure experiments were also performed. Exposed plants were photographed during the experiment and images were analyzed to calculate root length and leaf areas. Arabidopsis total RNA was extracted and Quantitative Real Time-PCR (qPCR) analyses were performed on gene expression of CRUCIFERIN 3 (CRU3), copper transport protein1 (COTP1), Redox Responsive Transcription Factor1 (RRTF1), Fe Superoxide Dismutase 1, (FSD1), Catalase3 (CAT3), Thylakoidal Ascorbate Peroxidase (TAPX), a cytosolic Ascorbate Peroxidase1 (APX1), and NADPH/respiratory burst oxidase protein D (RbohD). Four different reference genes were analysed to normalize the results of the qPCR. The best of the four genes was selected and the most stable gene for normalization was used. Our data show for the first time that reversing the GMF polarity using triaxial coils has significant effects on plant growth and gene expression. This supports the hypothesis that GMF reversal contributes to inducing changes in plant development that might justify a higher selective pressure, eventually leading to plant evolution.

The aim of this protocol is to provide a method to assess whether reversal of the geomagnetic field (GMF) may affect plant development and gene expression of Arabidopsis thaliana ecotype Col 0. Triaxial coils as shown in Figure 1A are used to reverse the GMF when set with the appropriate drive voltages (Figure 1B), obtained as described in step 1.5 in the protocol. The dimensions of the triaxial coils are ~2 x 2 x 2 m3, which allowed sufficient space with reversed GMF conditions to host several Petri plates. Controls were grown in the same environmental conditions and at normal GMF values. After 10 days of exposure to normal and reversed GMF conditions, the phenotype of plants showed evident morphological alterations. As shown in Figure 2, control plants (i.e., grown in normal GMF conditions) showed root lengths with significantly (Dunn-Sidak and Bonferroni Adjusted Prob &#60;0.001; Student&#8217;s t = 10.68, df = 31) higher values (29.41 mm; SEM = 1.04; N = 32) with respect to plants exposed to reversed GMF (17.53 mm; SEM = 0.58; N = 36). In GMF-reversed plants, the morphology of shoots was also altered by showing a reduced development of leaflet expansion. Plants exposed to normal conditions showed an average leaf area of 4.95 mm2 (SEM 0.025, N = 54), whereas plants exposed to reversed GMF conditions showed significantly (Dunn-Sidak and Bonferroni Adjusted Prob = &#60;0.001; student&#8217;s t = 31.32, df = 53) lower leaf area values (3.71 mm2; SEM = 0.032; N = 54). Therefore, exposure of Arabidopsis to reversed GMF conditions induced a reduction in both root length and leaf area.
Leaf expansion and root growth are dependent on both the division and the elongation of cells 27. Therefore, plant development, productivity and overall fitness are dependent on an optimal shoot- and root-system architecture 28. The reduced root length and leaf size of plants exposed to reversed GMF conditions indicate the presence of a sensing system able to not only perceive variations in magnetic field intensity, but also to respond to changes in the magnetic field &#8220;direction&#8221; compared to gravity. The hypothesis that GMF reversal may affect plant growth finds compelling evidence in our experiments, which demonstrate that GMF reversal conditions can significantly affect plant development.
The morphological changes were also accompanied by changes in gene expression. Among housekeeping genes, the most stable gene was the elongationfactor1B alpha-subunit 2. The first group of genes (CRU3, COTP1, RRTF1) showed a dramatic alteration in the gene expression (Figure 3). Shoot expression of all three genes was significantly increased (P &#60;0.05) by about 2.5-fold in plants exposed to reserved GMF conditions. Root expression of CRU3 was upregulated in the roots in plants exposed to normal GMF conditions, but was significantly (P &#60;0.05) downregulated in reversed GMF conditions. The opposite was found for COTP1 and RRTF1, which were downregulated in normal conditions and upregulated in the presence of GMF reversal (Figure 3).
Cruciferin (a 12 S globulin) is the most abundant storage protein in the seeds of A. thaliana and other crucifers and is synthesized as a precursor in the rough endoplasmic reticulum. It is then transported to the protein storage vacuoles 13. Seedling germination requires the breakdown of cruciferin, which is used as an initial source of nitrogen. Down-regulation of cruciferin degradation reduces embryos development by impairing cell structures or cell components development 29,30. Our results show that upregulation of CRU3 correlates with a lower leaf expansion and a reduced root length, thus indicating that this gene in sensitive to GMF reversal and that its overexpression may contribute to the reduction of plant development. Moreover, GMF reversal induces a significant downregulation of CRU3 in roots, which correlates with a reduced root length. Copper is an essential cofactor for key processes in plants, but it exerts harmful effects when in excess; thus, overexpressing copper transport compromises plant growth. The effect of GMF reversal was a significant overexpression of COTP1 in both shoots and roots, thus explaining the reduced plant growth. Ion stress impairs chloroplast metabolism, which is tightly linked to the redox state of the cell. In Arabidopsis the transcription factor RRTF1 is important for the expression of genes associated to the ability to adjust to redox changes 31. Therefore, when plants are exposed to external stimuli able to alter their physiological and developmental programs an overexpression of this important transcription factor is expected. Reversal of the GMF induced a significant overexpression of RRTF1 in both shoots and roots, thus indicating higher oxidative stress responses of plants to reversed GMF conditions.
Interesting results are obtained by analyzing the five genes involved in oxidative stress. In general, all genes extracted and analyzed in shoots did not show significant differences (P &#62;0.05) when plants were grown in normal or reversed GMF conditions (Figure 4 and Figure 5). However, a significant down-regulation was always observed in roots of plants exposed to reversed GMF conditions. In particular, CAT3 showed the highest downregulation (Figure 5), followed in order of downregulation by APX1, FSD1, RBOHD and TAPX (Figure 4).
Cross tolerance to abiotic and biotic stress is provided by the activation of different genes involved in several biochemical pathways. RRTF1 transcription factor facilitates the synergistic co-activation of gene expression of these pathways 15,31, and can be potentially involved in oxidative stress 32. Therefore, upregulation of RRTF1 is expected when oxygen scavenging is reduced. Downregulation of root scavenging enzymes correlates with the upregulation of RRTF1, which acts in response to increased oxidative stress. The dramatic root downregulation of CAT3, APX1 and TAPX indicates the reduced ability of root cells to scavenge H2O2, which is accompanied by the reduced ability to dismutate the superoxide anion by downregulation of FSD1. The oxidative stress responses is higher in roots, which appear to be the main site of reversed GMF perception.
<img alt="Figure 1" src="/files/ftp_upload/53286/53286fig1.jpg" /> 
Figure 1.&#160;Geomagnetic field Compensation system. (A) triaxial coils (comprising a pair of octagonal coils for each of three perpendicular axes) used to reverse the geomagnetic field vector. (B) A computer-controlled power supply is connected to each pair of Helmholtz coils. (Voltages in these figures are arbitrary) <a href="https://www.jove.com/files/ftp_upload/53286/53286fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53286/53286fig2.jpg" /> 
Figure 2.&#160;Effects of geomagnetic field reversal on Arabidopsis morphology. After ten days of exposure, control plants (i.e., those exposed to normal GMF conditions) show a significantly greater root length and more expanded leaflets compared to plants that were exposed to reversed GMF conditions. Metric bar = 18 mm. <a href="https://www.jove.com/files/ftp_upload/53286/53286fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53286/53286fig3.jpg" /> 
Figure 3.&#160;Effects of geomagnetic field reversal on Arabidopsis gene expression. After ten days of exposure, total RNA of control and treated plants was extracted and analysed by Real-Time PCR for expression analysis. The effect of reversal of the GMF was to induce a drastic change in the gene expression of all genes that were tested. CRU3, Cruciferin 3; COTP1, Copper Transport Protein1; RRTF1, Redox Responsive Transcription Factor1. Bars indicate standard error; asterisks indicates significant (P &#60;0.05) differences between plants exposed to reversed and normal GMF conditions. <a href="https://www.jove.com/files/ftp_upload/53286/53286fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53286/53286fig4.jpg" /> 
Figure 4.&#160;Effects of geomagnetic field reversal on Arabidopsis antioxidant-related gene expression. After ten days of exposure, total RNA of control and treated plants is isolated and employed for gene expression analysis using Real-Time PCR. The effect of reversal of the GMF was to induce no significant changes in shoot gene expression; however, a drastic downregulation was observed in root gene expression of plants grown under reversed GMF conditions. TAPX, Thylakoidal Ascorbate Peroxidase; APX1, Ascorbate Peroxidase1; FSD1, Fe Superoxide Dismutase1; RbohD, NADPH/Respiratory burst oxidase protein D. Bars indicate standard error; asterisks indicates significant (P &#60;0.05) differences between plants exposed to reversed and normal GMF conditions. <a href="https://www.jove.com/files/ftp_upload/53286/53286fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53286/53286fig5.jpg" /> 
Figure 5. Effects of the geomagnetic field reversal on Arabidopsis Catalase 3 (CAT3) gene expression. After ten days of exposure, total RNA of control and treated plants is isolated and employed for gene expression analysis using Real-Time PCR. The effect of reversal of the GMF was to induce no significant changes in shoot gene expression; however, a drastic downregulation was observed in root gene expression of plants grown under reversed GMF conditions. Bars indicate standard error; asterisks indicates significant (P &#60;0.05) differences between plants exposed to reversed and normal GMF conditions. <a href="https://www.jove.com/files/ftp_upload/53286/53286fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression at JoVE.com

Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

This protocol describes a method to test the hypothesis that the reversal of the effective geomagnetic field (GMF) induces differential gene expression and alters the morphology of Arabidopsis thaliana. A triaxial octagonal system of Helmholtz coil-pairs was used to artificially generate reversed GMF conditions in the plant growth chamber.

Geomagnetic Field (Gmf) and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) ...

geomagnetic-field-gmf-plant-evolution-investigating-effects-gmf

Research

JoVE Journal

Developmental Biology

13.3K Views.  University of Turin. This protocol describes a method to test the hypothesis that the reversal of the effective geomagnetic field (GMF) induces differential gene expression and alters the morphology of Arabidopsis thaliana. A triaxial octagonal system of Helmholtz coil-pairs was used to artificially generate reversed GMF conditions in the plant growth chamber.

Video: Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (&#946;-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

Genetics

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Biology

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Geomagnetic Field (Gmf) and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

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